IN VITRO REPLICATION OF HEPATITIS C VIRUS

HEPATITIS C VIRUS IS A POSITIVE STRANDED RNA VIRUS WHICH IS CURRENTLY RECOGNIZED AS THE MAJOR CAUSATIVE AGENT OF NON-A, NON-B HEPATITIS. SINCE ITS DISCOVERY TWO YEARS AGO, LITTLE PROGRESS HAS BEEN MADE TOWARD INCREASING OUR UNDERSTANDING OF THE MOLECULAR EVENTS REGULATING VIRAL REPLICATION. THIS HAS BEEN PRIMARILY DUE TO THE FACT THAT ATTEMPTS TO PROPAGATE HCV IN VITRO HAVE NOT BEEN SUCCESSFUL. WE PLAN TO OBTAIN REPLICATION OF HCV IN VITRO BY CONSTRUCTING A FULL-LENGTH CDNA CLONE FROM WHICH POTENTIALLY INFECTIOUS RNA WILL BE TRANSCRIBED. TRANSFECTIONOF THIS RNA INTO A PERMISSIVE CELL LINE LEADS TO TRANSLATION OF STRUCTURAL AND NON-STRUCTURAL PROTEINS AND VIRAL REPLICATION. REPLICATION WILL BE MONITORED BY A SENSITIVE AND SPECIFIC EIA WHICH HAS BEEN DEVELOPED TO DETECT THE HCV CAPSID PROTEIN P22, OR BY STRAND SPECIFIC PCR TO DETECT THE SYNTHESIS OF COMPLIMENTARY MINUS STRAND INTERMEDIATES. DEVELOPMENT OF AN ASSAY FOR HCV REPLICATION COULD LEAD TO THE ISOLATION OF SPECIFIC VIRAL INHIBITORS WHICH WOULD PROVE USEFUL IN THE TREATMENT OF PATIENTS WITH CHRONIC NON-A, NON-B HEPATITIS. FURTHERMORE, SYNTHESIS OF AN INFECTIOUS CDNA CLONE WOULD ENABLE US TO INTRODUCE SPECIFIC MUTATIONS INTO THE GENOME TO DETERMINE THE FUNCTION OF HCV PROTEINS, OR TO CREATE AN ATTENUATED STRAIN FOR VACCINEDEVELOPMENT.